New Method for Detecting the Suppressing Effect of Enzyme Activity by Aminopeptidase N Inhibitor

Aminopeptidase N, also known as CD13, is a transmembrance protease with many functions. CD13 is involved in inflammatory diseases and cancers. A convenient and reliable laboratory test method for detecting the suppressing effects of enzyme activity would be useful for study of CD13 inhibitors. Porcine CD13 (pCD13) was traditionally considered an enzyme source but has significant practical disadvantages. pCD13 is not a human source, and the accuracy and reliability of experimental results are greatly reduced. In this study, a modified detection method with K562-CD13 monoclonal cells, a human-derived cell line, was established to detect the suppressing effects of enzyme activity by the CD13 inhibitor. In this method, K562-CD13 monoclonal cells were used as enzyme source and L-leucine p-nitroaniline hydrochloride as substrate. Using CD13 enzyme activity analyses, we found that the ability of the catalytic substrate was weaker in K562 cells than in the other cell lines, and K562-CD13 cells expressed significantly higher levels of CD13 enzyme activity than parental K562 cells. The enzyme activity of CD13 was detected with the new method after ubenimex treatment. The enzyme activity was significantly inhibited by ubenimex in a dose-dependent manner. In summary, this study proposes a sensitive, stable, and objective laboratory method for detecting the inhibitory effect of the CD13 inhibitor.